Resistance to antimicrobial chemotherapies is a major contributor to morbidity, mortality, and rising healthcare costs. The prevalence of drug-resistant pathogens against conventional antibiotics has been rising over the last several decades. Of great concern is methicillin-resistant Staphylococcus aureus (MRSA), which is rapidly diminishing the available treatment options for S. aureus infections. As drug resistances continue to emerge there is an urgent need to develop alternative therapeutic approaches such as the development of adjunctive therapies to potentiate the activity of existing antimicrobials. Recently we have discovered that amidase activity of cell wall modifying enzymes (named SsaA1 and SsaA2) mediates S. aureus biofilm resistance to the conventional antibiotic fusidic acid. That is, genetic inactivation of ssaA1 or ssaA2 results in sensitivity of biofilms to fusidic acid. This result suggests that small molecules capable of inhibiting SsaA amidase activity could be used as an adjunctive in conjunction with fusidic acid to effectively eliminate S. aureus biofilms. Mutants in ssaA1 also displayed reduced frequency of fusidic acid resistance emergence, suggesting that in addition to rendering biofilm bacteria sensitive to an antimicrobial, small molecule SsaA amidase inhibitors would also reduce the emergence of drug resistant bacteria. SsaA amidases are an ideal target for small molecule therapies because they are located on the outside of cells making them readily accessible and because humans lack peptidoglycan and the associated biosynthetic machinery (including SsaA amidase homologous). The primary goal of this R21 research is to identify chemical inhibitors of SsaA amidase activity and characterize these molecules for use as potential adjunctive therapeutics. To achieve the goal, we propose the following aims: (1) Identify chemical compounds that eliminate SsaA amidase activity via high throughput screening and (2) Elucidate the ability of SsaA chemical inhibitors to sensitize Staphylococcus biofilms to antimicrobial treatment in in vitro and in vivo models of biofilm development. During the next phase of research (R33), we plan on translating our basic findings for clinical applications by investigating chemical optimization of lead compounds, characterizing antimicrobial activity against other gram-positive pathogens, and developing formulation/delivery systems to be tested with in vivo animal models. The PI's experience in S. aureus biology and the strength of the University of Michigan's Center for Chemical Genomics uniquely position us to conduct this research and make profound discoveries.